Study On Glucose Isomerase From Alicyclobacillus Sp.A4 And Caldicellulosiruptor Bescii

High fructose corn syrup（HFCS）made from corn starch is an important sweetener.Due to the advantages of sweetness,low cost and high solubility,HFCS is used extensively in various industrial fields,including the food,detergent,and pharmaceutics.According to the fructose content,HFCS is divided into three types:HFCS-42,HFCS-55 and HFCS-90.Glucose isomerase（GI）that catalyzes the conversion of D-glucose to D-fructose is one of the most important industrial enzymes for the production of high fructose corn syrup.Due to the low efficiency of commercial glucose isomerase,the current industrial production of HFCS is mainly the HFCS-42.Although HFCS-55 with better taste and sweetness has larger market demand than HFCS-42.its commercial production requires complex processes including chromatography,purification and concentration.Both improving the conversion rate of D-glucose to D-fructose and increasing the substrate concentration（glucose）are effective ways to reduce the production cost.It has been reported that a thermodynamic equilibriumb exists between the isomerization reaction of glucose and fructose.Along with the increase of reaction temperature,the conversion rate of glucose to fructose also gradually increases.Thus a thermostable GI is more favorable for the one-step production of HFCS-55.In the present study,two GI were cloned from Alicyclobacillus sp.A4 and Caldicellulosiruptor bescii,and expressed in Escherichia coli BL21（DE3）.After the protein was purified by Hig-tag magnetic beads,its enzymatic properties and conversion rate were determined in detail.The optimum temperature and optimum pH of A4GI were65°C and pH 7.5,and it was stable below 55°C and over pH 6.0-11.0.The K_m and V_maxax values of A4GI were 99.8 mM and 3.75 U/mg.In addition,the thermal stability of A4GI was modified and the mutant D109K was designed using site-directed mutagenesis.Compared to the wild type,the thermal stability of the mutants was improved.In comparison to A4GI,CbGI exhibited superior enzymatic properties and greater conversion efficiency.The optimal temperature and optimum pH of CbGI were 80°C and pH 7.5,and it was stable at pH 5.0-11.0 and below 85°C.Kinetic measurements showed that the K_m and V_max values of CbGI were 42.61 mM and 8.22 U/mg,respectively.In comparison with other reported GIs,CbGI exhibited higher substrate affinity.In the conversion assay,The result showed that the conversion rate of A4GI and CbGI increased along with the increased concentration of D-glucose.The maximum conversion rate of CbGI and A4GI were 57.3%and 52.7%respectively when 3 M D-glucose was used as the substrate（w/v=54%）.In conclusion,CbGI with high temperature,wide pH,and high conversion rate at high concentrations of substrates have the potential for industrial application of HFCS-55.